WO2024072241A1 - Compound - diagnostic marker for ovarian cancer, method for detecting enzymatic activity, method for diagnosis of ovarian cancer, kit comprising the compound, uses of the compound and method for the treatment of ovarian cancer - Google Patents
Compound - diagnostic marker for ovarian cancer, method for detecting enzymatic activity, method for diagnosis of ovarian cancer, kit comprising the compound, uses of the compound and method for the treatment of ovarian cancer Download PDFInfo
- Publication number
- WO2024072241A1 WO2024072241A1 PCT/PL2023/050081 PL2023050081W WO2024072241A1 WO 2024072241 A1 WO2024072241 A1 WO 2024072241A1 PL 2023050081 W PL2023050081 W PL 2023050081W WO 2024072241 A1 WO2024072241 A1 WO 2024072241A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- formula
- ovarian cancer
- abz
- phe
- Prior art date
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 181
- 206010033128 Ovarian cancer Diseases 0.000 title claims abstract description 147
- 206010061535 Ovarian neoplasm Diseases 0.000 title claims abstract description 143
- 238000000034 method Methods 0.000 title claims abstract description 96
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 67
- 238000003745 diagnosis Methods 0.000 title claims abstract description 43
- 239000003550 marker Substances 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 27
- 210000001124 body fluid Anatomy 0.000 claims abstract description 23
- 239000010839 body fluid Substances 0.000 claims abstract description 23
- 238000000338 in vitro Methods 0.000 claims abstract description 9
- 239000012634 fragment Substances 0.000 claims description 35
- 210000002700 urine Anatomy 0.000 claims description 35
- 238000003776 cleavage reaction Methods 0.000 claims description 28
- 230000007017 scission Effects 0.000 claims description 28
- 238000005259 measurement Methods 0.000 claims description 25
- 241000282414 Homo sapiens Species 0.000 claims description 20
- 238000002835 absorbance Methods 0.000 claims description 14
- 230000003287 optical effect Effects 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 13
- 230000003301 hydrolyzing effect Effects 0.000 claims description 10
- 230000002797 proteolythic effect Effects 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 10
- KZZWQCKYLNIOBT-UHFFFAOYSA-N 5-amino-2-nitrobenzoic acid Chemical compound NC1=CC=C([N+]([O-])=O)C(C(O)=O)=C1 KZZWQCKYLNIOBT-UHFFFAOYSA-N 0.000 claims description 9
- 208000007660 Residual Neoplasm Diseases 0.000 claims description 9
- 238000002271 resection Methods 0.000 claims description 6
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 3
- SJQRQOKXQKVJGJ-UHFFFAOYSA-N 5-(2-aminoethylamino)naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(NCCN)=CC=CC2=C1S(O)(=O)=O SJQRQOKXQKVJGJ-UHFFFAOYSA-N 0.000 claims description 3
- XPDXVDYUQZHFPV-UHFFFAOYSA-N Dansyl Chloride Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(Cl)(=O)=O XPDXVDYUQZHFPV-UHFFFAOYSA-N 0.000 claims description 3
- AIUKPEQJKQUQKZ-UHFFFAOYSA-N n'-(2,4-dinitrophenyl)ethane-1,2-diamine Chemical compound NCCNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O AIUKPEQJKQUQKZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 33
- 201000011510 cancer Diseases 0.000 abstract description 27
- 239000003814 drug Substances 0.000 abstract description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 58
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 57
- 239000011347 resin Substances 0.000 description 48
- 229920005989 resin Polymers 0.000 description 48
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 39
- 238000012360 testing method Methods 0.000 description 27
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 24
- 108090000765 processed proteins & peptides Proteins 0.000 description 23
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 18
- 150000003862 amino acid derivatives Chemical class 0.000 description 17
- 230000015572 biosynthetic process Effects 0.000 description 16
- 230000008569 process Effects 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 238000003786 synthesis reaction Methods 0.000 description 16
- 239000002904 solvent Substances 0.000 description 14
- UGNWTBMOAKPKBL-UHFFFAOYSA-N tetrachloro-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(Cl)=C(Cl)C1=O UGNWTBMOAKPKBL-UHFFFAOYSA-N 0.000 description 13
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 11
- 125000000539 amino acid group Chemical group 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- 150000001408 amides Chemical class 0.000 description 10
- 230000008021 deposition Effects 0.000 description 10
- 239000000758 substrate Substances 0.000 description 9
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 8
- 230000010933 acylation Effects 0.000 description 8
- 238000005917 acylation reaction Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 101000955067 Homo sapiens WAP four-disulfide core domain protein 2 Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 6
- 101100074988 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) nmp-1 gene Proteins 0.000 description 6
- 102100038965 WAP four-disulfide core domain protein 2 Human genes 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 6
- 238000002405 diagnostic procedure Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- AHDSRXYHVZECER-UHFFFAOYSA-N 2,4,6-tris[(dimethylamino)methyl]phenol Chemical compound CN(C)CC1=CC(CN(C)C)=C(O)C(CN(C)C)=C1 AHDSRXYHVZECER-UHFFFAOYSA-N 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 210000001672 ovary Anatomy 0.000 description 5
- 125000006239 protecting group Chemical group 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 239000012317 TBTU Substances 0.000 description 4
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 208000007571 Ovarian Epithelial Carcinoma Diseases 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 230000010534 mechanism of action Effects 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 239000000439 tumor marker Substances 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- LZOLWEQBVPVDPR-VLIAUNLRSA-N (2s,3r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@H](OC(C)(C)C)C)C(O)=O)C3=CC=CC=C3C2=C1 LZOLWEQBVPVDPR-VLIAUNLRSA-N 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- -1 9-fluorenylmethoxy carbonyl Chemical group 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- GXHDGYOXPNQCKM-XVSYOHENSA-N Asp-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O GXHDGYOXPNQCKM-XVSYOHENSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 239000000104 diagnostic biomarker Substances 0.000 description 2
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000012454 non-polar solvent Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 1
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 1
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 1
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 1
- BYGHHEDJDSLEKK-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]benzoic acid Chemical compound CC(C)(C)OC(=O)NC1=CC=CC=C1C(O)=O BYGHHEDJDSLEKK-UHFFFAOYSA-N 0.000 description 1
- 206010000084 Abdominal pain lower Diseases 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 101100423701 Arabidopsis thaliana OVA1 gene Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 206010061336 Pelvic neoplasm Diseases 0.000 description 1
- 108010071690 Prealbumin Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102000009190 Transthyretin Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 201000003908 endometrial adenocarcinoma Diseases 0.000 description 1
- 208000029382 endometrium adenocarcinoma Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 230000005906 menstruation Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- LQRJAEQXMSMEDP-XCHBZYMASA-N peptide a Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)NCCCC[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C/C=1C=CC=CC=1)C(N)=O)C(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C\C1=CC=CC=C1 LQRJAEQXMSMEDP-XCHBZYMASA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Definitions
- Compound - diagnostic marker for ovarian cancer for ovarian cancer, method for detecting enzymatic activity, method for diagnosis of ovarian cancer, kit comprising the compound, uses of the compound and method for the treatment of ovarian cancer
- the invention relates to a new chemical compound, a diagnostic marker, for use in medicine, more specifically in cancer diagnostics, in particular diagnosis of ovarian cancer.
- the invention also relates to an m vitro method for detecting enzymatic activity present in a subject’s body fluid, in particular deriving from ovarian cancer cells, using the compound, an in vitro method for the diagnosis of ovarian cancer using the compound, a kit comprising the compound, use of the compound for the detection of enzymatic activity specific to ovarian cancer, use of the compound for the diagnosis of ovarian cancer, the compound for use as a diagnostic marker for ovarian cancer.
- the invention further relates to a method for the treatment of ovarian cancer comprising a step of carrying out the method for the diagnosis of ovarian cancer as specified above.
- Ovarian cancer is the 8th most common cancer diagnosed in women and the third cancer of the female reproductive organs. More than 300 000 new cases were reported in 2020. More than 114 000 women with ovarian cancer die each year worldwide. Risk factors for this cancer include childlessness, early or late menstruation, late menopause (after the age of 55), hormone therapy and active or passive smoking. In its early stages, ovarian cancer usually does not produce any symptoms; it is only when the disease is more advanced that gastrointestinal symptoms appear. Despite its name, ovarian cancer is not only a disease of the ovaries themselves - in most cases, so-called peritoneal spread is observed, i.e. the presence of tumours on other abdominal organs.
- Symptoms of ovarian cancer are not specific and are usually of a general nature such as ascites, lower abdominal pain, bleeding from the reproductive tract.
- the diagnosis is confirmed by the result of the patient's gynaecological examination, ultrasound examination and the CA-125 marker present in the blood.
- Ca-125 is useful in the diagnosis, monitoring and prognosis of the course of the disease and the recognition of its recurrence. In about half of patients with early-stage ovarian cancer, it manifests itself with an elevated CA-125 level. It is estimated that more than 80 per cent of women with ovarian cancer have an elevated CA-125 marker level.
- CA-125 The sensitivity and specificity of CA-125 in predicting the occurrence of malignant tumours, as well as its positive predictive value, are low, since before surgery, CA-125 levels are normal in about 50 per cent of patients with stage I epithelial ovarian cancer. In addition, increased CA-125 levels are influenced by a number of conditions, including the presence of malignancies of other abdominal organs and many benign conditions. Attempts are being made to use CA-125 as a predictive marker, but the use of this marker for diagnostic tests for the detection, including early, ovarian cancer is not recommended due to the low selectivity of this test.
- OVA1 A new qualitative serum test to help predict the diagnosis of ovarian cancer in women with pelvic tumours has been released for sale. This test, known as OVA1, is based on the assessment of five biological markers: transthyretin, apolipoprotein A-l, microglobulin P2, transferrin and CA-125.
- HE4 human epididymis protein 4 from epithelial cells, human epididymis protein 4
- HE4 is a protease inhibitor found in malignant epithelial ovarian cancer cells. It can be detected in the serum of women with ovarian cancer.
- the combined determination of HE4 and CA-125 has diagnostic parameters superior to both results interpreted alone.
- the ROMA Read of ovarian malignancy algorithm
- an algorithm for assessing the risk of the presence of epithelial ovarian cancer and the likelihood that an existing small pelvic lesion of unclear status is malignant was created.
- CA-125 and HE4 the ROMA (Risk of ovarian malignancy algorithm)
- chromogenic peptide molecules that undergo enzymatic breakdown into smaller fragments resulting in a change or increase in the colour of the solution being tested. This chromogenic effect is a consequence of the release of a chromophore (e.g. 4-nitroanilide or 2-aminobenzoic acid) from a chromogenic peptide molecule.
- a chromophore e.g. 4-nitroanilide or 2-aminobenzoic acid
- chromogenic peptides which consist in attaching individual components in appropriate time and stoichiometric conditions are also known in the prior art.
- the process of attaching consists of subsequent steps in which individual elements (amino acid derivatives) are attached, residues are washed off and protecting groups are sequentially removed and washed again. This cycle is repeated for each amino acid residue.
- the obtained peptide is separated from resin by a reaction in acidic conditions. Subsequently, the solution is separated from resin in the filtration process and then the peptide is precipitated from the solution by means of a non-polar solvent.
- Chromogenic peptide compounds appropriate for a specific and early diagnosis of ovarian cancer or methods to obtain them are however not known in the prior art.
- the object of the present invention is to provide a novel, specific diagnostic marker for ovarian cancer and diagnostic methods using such a marker for a non-invasive, quick, sensitive and specific, early detection of ovarian cancer, which would also be appropriate for screening tests, as well as treatment methods using such a marker.
- the invention provides a compound having formula 1 :
- Xl 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (formula 1), wherein XI comprises or consists of molecule Cl, and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of florescence donor and fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into the fragments Xl-Asp-Thr- Phe-Ile-OH (Fragment 1) and X2 (Fragment 2) with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
- the compound according to the invention preferably undergoes hydrolytic cleavage, more preferably proteolytic.
- the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ)/ 5-amino-2-nitrobenzoic (ANB), (ABZ)/pNA, ABZ/ANB-NH 2 , ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/TyrQ-NCh), more preferably the pair of Cl and C2 is (ABZ)/pNA or ABZ/ANB-NH2.
- the compound according to the invention is a compound having formula 2: ABZ- Asp-Thr-Phe-Ile-ANB-NFF (formula 2) or a compound having formula 3: ABZ-Asp-Thr- Phe-Ile-pNA (formula 3).
- the compound according to the invention undergoes hydrolytic cleavage with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2.
- the invention further provides an in vitro method for detecting enzymatic activity present in a subject’s body fluid, in particular deriving from ovarian cancer cells, comprising: a) contacting a sample of body fluid with the compound having formula 1 : Xl 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (formula 1), wherein XI comprises or consists of molecule Cl and X2 comprises or consist of molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the said compound undergoes enzymatic cleavage into the fragments Xl-Asp- Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), and b) detecting a measurable optical signal which is generated upon spatial separation of molecules Cl and C2.
- enzymatic activity is preferably hydrolytic activity, more preferably proteolytic activity.
- the compound having formula 2 ABZ-Asp-Thr-Phe-Ile-ANB-NHz (formula 2) or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is preferably used.
- a urine more preferably human urine
- the invention also relates to an in vitro method for the diagnosis of ovarian cancer, wherein the presence or absence of ovarian cancer in a subject is detected by measuring enzymatic activity specific to ovarian cancer in a body fluid sample from an examined subject, and wherein the absence of the said enzymatic activity indicates the absence of ovarian cancer, whereas the presence of the said enzymatic activity indicates the presence of ovarian cancer.
- the detection of enzymatic activity is carried out by the method for detecting enzymatic activity as defined above.
- the measurement of the said enzymatic activity is performed using the compound having formula 1 :
- Xl 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (formula 1), wherein XI comprises or consists of molecule Cl and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the said compound undergoes enzymatic cleavage into the fragments Xl-Asp- Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) with the generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
- the said body fluid sample is preferably incubated with the said compound in a measurement buffer having neutral or alkaline pH, more preferably physiological, within the range of sample-to- measurement buffer ratio of from 1 :2 to 1 : 10, preferably 1 :5.
- the said compound is preferably used at a concentration of 0.1-10 mg/mL, in particular 0.25-7.5 mg/mL.
- the compound having formula 2 ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (formula 2) or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is preferably used.
- a urine sample more preferably human urine, is preferably used.
- the measurement of the said enzymatic activity preferably comprises the measurement of absorbance intensity within the range of 300-500 nm, more preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, more preferably 36-38° C.
- the invention further provides a kit comprising any compound according to the invention as defined above and a measurement buffer.
- the said compound is preferably the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp- Thr-Phe-Ile-pNA.
- the invention also provides the use of any compound according to the invention as defined above for the detection of enzymatic activity specific to ovarian cancer.
- the invention also provides the use of any compound according to the invention as defined above for the diagnosis of ovarian cancer.
- the diagnosis of ovarian cancer comprises the detection of primary ovarian cancer, detection of Minimal Residual Disease after surgical resection of ovarian cancer and/or detection of ovarian cancer recurrence.
- the compound in the uses according to the invention is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NJL or a compound having formula 3: ABZ-Asp- Thr-Phe-Ile-pNA.
- the invention further provides any of the compounds according to the invention as defined above for use as a diagnostic marker for the detection of ovarian cancer.
- the compound for use as the diagnostic marker according to the invention is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFF or a compound having formula 3 : ABZ-Asp-Thr-Phe-Ile-pNA.
- the invention further provides a method for the treatment of ovarian cancer, wherein a) the presence of enzymatic activity specific to ovarian cancer is detected by any method as defined above in a body fluid sample from an examined subject, and b) if the presence of the said enzymatic activity is found in the said sample, a treatment for ovarian cancer is applied in the subject.
- the said enzymatic activity specific to ovarian cancer is monitored at predetermined time intervals.
- a urine sample preferably human urine
- the sample is used as the sample.
- the compound having formula 2 ABZ-Asp-Thr-Phe-Ile-ANB-NFF or a compound having formula 3: ABZ-Asp- Thr-Phe-Ile-pNAis used as the said compound.
- a chromogenic compound or a chromogenic molecule means a compound having chromogenic properties. Chromogenic properties mean the ability of a compound to form a coloured product.
- a fluorescent compound or a fluorescent molecule means a compound having fluorogenic properties. Fluorogenic properties mean the ability of a compound to form a product emitting fluorescence.
- NMP stands for A-methylpirrolidone
- DMF stands for dimethylformamide
- DCM stands for methylene chloride or dichloromethane
- pNA 4-nitroaniline or para-nitroaniline
- ABZ stands for 2-aminobenzoic acid
- ANB-NH2 stands for amide of 5-amino-2-nitrobenzoic acid
- Boc stands for tert-butyloxycarbonyl group
- Fmoc stands for 9- fluorenylometoxycarbonyl group
- TFA stands for trifluoroacetic acid.
- ovarian cancer shall be understood to mean primary cancer of ovary (malignant neoplasm) that develops from tissues located in the ovary.
- Ovarian cancer is most frequently endometrial adenocarcinoma (about 90 %), less frequently serous clear cell cancer.
- ovarian cancer as used herein comprises thus all malignant ovarian neoplasms which develop from the tissues located in the ovary.
- diagnosis of ovarian cancer shall be understood to mean identification of this disease, in particular at its early stage at which other diagnostic methods are not sensitive and/or specific enough.
- diagnosis of ovarian cancer also comprises the detection of Minimal Residual Disease (MRD) after surgical resection of ovarian cancer and detection of ovarian cancer recurrence after previously completed treatment of ovarian cancer.
- MRD Minimal Residual Disease
- treatment of ovarian cancer shall be understood to mean a treatment at an early stage of progression of the disease, which makes it possible to significantly prolong survival time and improve the quality of life of the diseased subjects.
- monitoring shall be understood to mean the diagnosing of Minimal Residual Disease (MRD)) - the presence of a small number of cancer cells that have survived in the organism (during treatment or remission), in the amounts undetectable by means of standard diagnostic methods.
- MRD Minimal Residual Disease
- subject shall be understood to mean a human subject or a mammal that is suspected to have ovarian cancer, or alternatively a human subject or a mammal belonging to a group with an increased risk of ovarian cancer, or a human subject or a mammal after resection of ovarian cancer or after a finished treatment of ovarian cancer.
- the subject is preferably a human subject.
- the compounds according to the invention have chromogenic properties due to the presence of a chromophore, and fluorogenic properties, i.e. they contain molecules of a fluorescence donor and acceptor.
- the examined subject is preferably a human subject.
- the body fluid is preferably urine, more preferably human urine.
- Xl 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (formula 1), wherein XI is an amino acid derivative or a peptide fragment comprising molecule Cl, or XI consists of such a molecule Cl, and X2 is an amino acid derivative or a peptide fragment comprising molecule C2, or X2 consists of such a molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor.
- the superscripts indicate subsequent positions of residues in the compound according to the invention and the sequence of attaching of the residues during synthesis.
- chemical formula 1 can be alternatively written without indicating the numbering of residues.
- the core of all compounds according to the invention is tetrapeptide with the indicated sequence of 4 amino acids, i.e. Asp-Thr-Phe-Ile (the notation in the three-letter amino acid abbreviation format equivalent to the notation: DTFI in the one-letter amino acid abbreviation format), which sequence is also presented in the Sequence Listing as sequence no. 1.
- the compound according to the invention undergoes enzymatic cleavage into the fragments: Xl-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) with the generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
- the measurable optical signal is measured by a method for measuring a change in absorbance/fluorescence after the enzymatic cleavage of the compound.
- molecules Cl and C2 are separated from each other by not more than 10 amino acid residues, which ensure efficient quenching of the fluorescence donor by the fluorescence acceptor. It is obvious for the skilled person that the key factor is the distance between the fluorescence donor and acceptor.
- the distance between molecules Cl and C2 can be greater than 10 amino acid residues.
- the compound due to its chromogenic properties and the presence of a reactive site at the position 5 enabling enzymatic (preferably proteolytic) cleavage into smaller fragments, is particularly suitable for use as a diagnostic marker, in particular a specific diagnostic biomarker for ovarian cancer, in particular for use in the early diagnosis of ovarian cancer.
- the compound according to the invention undergoes hydrolytic cleavage, more preferably proteolytic cleavage.
- the pair of molecules Cl and C2 is selected from a group consisting of: 2-aminobenzoic acid (ABZ)/5-amino-2-nitrobenzoic acid (ANB), (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO2), more preferably the pair of molecules Cl and C2 is ABZ/pNA or ABZ/ANB-NH 2 .
- the compound according to the invention is: the compound having formula 2:
- ABZ 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -pNA 6 (formula 3), wherein ABZ stands for 2-aminobenzoic acid, ANB-NH2 stands for amide of 5-amino-2- nitrobenzoic acid, and pNA stands for 4-nitroaniline.
- the compound is subject to hydrolytic cleavage with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2 in the case of the compound having formula 2, whereas in the case of the compound having formula 3, with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: pNA.
- fragments 2 are free chromophores.
- Spatial separation of molecules Cl and C2 being a result of the enzymatic cleavage of the compound according to the invention causes generation of a measurable optical signal because fluorescence emitted from the fluorescence donor is no longer quenched by the fluorescence acceptor.
- measurable optical signal can be detected preferably at a wavelength of 300-500 nm, more preferably 380-430 nm.
- the compounds according to the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides which consists in carrying out the process on a solid support in the form of a resin having an Fmoc group, which is removed in the course of the reaction.
- it can be an amide resin, e.g.
- Teenage S RAM or RinkAmide but any other commercially available resin can also be used.
- the resin used to carry out the process should be properly prepared.
- the preparation of the resin consists in increasing its volume by repeated washing with hydrophobic solvents.
- a resin with a deposition of 0.23 mmol/g is used.
- the Fmoc protecting group must be removed from the resin by washing it with a 20% solvent solution.
- the known processes for obtaining chromogenic peptides comprise attaching individual components in appropriate time and stoichiometric conditions.
- the attaching process consists of subsequent steps in which individual elements (amino acid derivatives) are attached, residuals are washed off and protecting groups are successively removed and washed again. This cycle is repeated for each amino acid residue.
- the obtained peptide is separated from the resin by a reaction under acidic conditions.
- the solution is separated from the resin in the filtration process and then the peptide is precipitated from the obtained solution by means of a non-polar solvent.
- the peptide precipitate obtained in this way is centrifuged.
- the method for the synthesis of the compound according to the invention consists in that the process is carried out on a solid support in the form of a resin, preferably having an Fmoc group, wherein before the start of the process the solid support is prepared by increasing its volume by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removing the Fmoc protecting group, preferably by washing with 10 - 30 % piperidine solution, in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone.
- hydrophobic solvents preferably dimethylformamide, methylene chloride or N-methylpyrrolidone
- the second aspect of the present invention provides an n vitro method for detecting enzymatic, preferably proteolytic, activity, present in a subject’s body fluid, in particular deriving from ovarian cancer cells, the method comprising a) contacting the body fluid sample with the compound according to the invention and b) detecting a measurable optical signal which is generated upon spatial separation of molecules Cl and C2 present in the compound according to the invention.
- the examined subject in this case is a human subject.
- the body fluid is urine, in particular human urine.
- the compound having formula 2 ABZ-Asp-Thr-Phe-Ile-ANB-NHz or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA is used.
- the third aspect of the present invention provides an in vitro method for the diagnosis of ovarian cancer in which the presence or absence or ovarian cancer in a subject is detected by measuring enzymatic activity specific to ovarian cancer in a body fluid sample from the examined subject, wherein the absence of the said enzymatic activity indicates the absence of ovarian cancer whereas the presence of the said enzymatic activity indicates the presence of ovarian cancer.
- detection of enzymatic activity is preferably carried out using methods for detecting enzymatic activity as discussed above.
- the subject is a human subject.
- the body fluid is urine, in particular human urine.
- the enzymatic activity specific to ovarian cancer is proteolytic activity.
- the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA is used.
- the measurement of the said enzymatic activity in the methods according to the invention comprises the measurement of absorbance intensity within the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, preferably 36- 38° C.
- the measurement of the said enzymatic activity is performed using the compound according to the invention in the range of concentrations of 0.1 - 10 mg/mL, more preferably at the concentration of 1 mg/mL.
- the tested sample is incubated with the compound according to the invention in a measurement buffer having a neutral or alkaline pH, preferably physiological, with a body fluid sample, preferably human urine, with the sample (e.g. of urine) to measurement buffer ratio ranging from 1 :2 to 1 :10, preferably 1 :5.
- the sample is preferably taken from a subject with a referral for the diagnosis of ovarian cancer.
- absorbance intensity is measured within the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, preferably 36-38° C.
- a maximally intensive measurable optical signal is obtained resulting from an increase in absorbance or fluorescence.
- the present invention provides a kit comprising any compound according to the invention and a measurement buffer.
- Measurement buffers are known in this art and a buffer suitable for use in the kit according to the invention is, for example, but without limitation, the Tris-HCl buffer.
- the said compound in the kit according to the invention is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.
- the present invention provides use of the compound according to the invention for the detection of enzymatic activity specific to ovarian cancer.
- the present invention provides use of the compound according to the invention for the diagnosis of ovarian cancer.
- the diagnosis of ovarian cancer comprises, according to the invention, the detection of primary ovarian cancer, detection of Minimal Residual Disease after surgical resection of ovarian cancer and/or detection of ovarian cancer recurrence after previously completed ovarian cancer treatment.
- the present invention provides the compound according to the invention for use as a diagnostic marker for the detection of ovarian cancer.
- the said compound is the compound having formula 2: ABZ- Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.
- the present invention provides a method for the treatment of ovarian cancer wherein a) the presence of enzymatic activity specific to ovarian cancer is detected by any method according to the invention as defined above, in a body fluid sample from the examined subject, and b) when the said enzymatic activity is found to be present in the said sample, a treatment of ovarian cancer is applied in the subject.
- the said enzymatic activity specific to ovarian cancer is monitored at predetermined time intervals as known in the art, e.g. every week, every several weeks, every month, every several months, every year or at any other intervals considered to be appropriate by the skilled person, in order to detect Minimal Residual Disease after surgical resection of ovarian cancer or recurrence.
- a urine sample preferably human urine, is used as the test sample.
- the compound having formula 2 ABZ-Asp- Thr-Phe-Ile-ANB-NHz (formula 2) or a compound having formula 3 : ABZ-Asp-Thr-Phe- Ile-pNA (formula 3) is used as the said compound.
- the advantages of the present invention consist in providing a novel chemical compound having properties that make it suitable for use for specific and sensitive detection of enzymatic activity specific to ovarian cancer, for use as a diagnostic biomarker for the detection of ovarian cancer, for use in a fast, non-invasive diagnosis of ovarian cancer, while enabling the detection of ovarian cancer at an early stage of its progression.
- Another advantage is that the diagnostic methods according to the invention can be successfully used in screening tests. This enables full diagnosis at an early stage of cancer progression and consequently a more effective treatment. Early diagnosis enables surgical treatment, which significantly prolongs patient’s survival time. It is also important when monitoring the effectiveness of the applied surgical treatment and/or chemotherapy of ovarian cancer since it is possible to detect Minimal Residual Disease or recurrence, if any.
- Fig. 1 shows the results of chromatographic analysis of the substrate cleavage, i.e. ABZ-Asp-Thr-Phe-Ile-ANB-NFE, in a sample of urine from a subject with ovarian cancer.
- Fig. 2 shows the rate of hydrolysis of the substrate - ABZ-Asp-Thr-Phe-Ile-ANB-NTL - in the samples of urine from subjects with diagnosed ovarian cancer (samples 1-20) and urine taken from healthy subjects (samples Z21 - Z40). Arabic numerals indicate the number of the selected urine sample.
- Fig. 3 shows the selectivity of hydrolysis of the substrate - ABZ 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 - ANB 6 -NH2 (i.e. compound of formula 2) in urine samples taken from subjects with diagnosed ovarian cancer (sample 1) and urine samples taken from subjects with the diagnosis of another neoplastic disease (samples 2 - 9).
- Arabic numerals indicate the numbers of given cancer types.
- the samples tested for each type of cancer were derived from 20 different patients for each of the tested cancers.
- the results are mean values for given cancer types.
- the results show selectivity of substrate cleavage in the case of urine from patients with ovarian cancer as compared to urine samples from patients suffering from other neoplasms.
- Fig. 4 shows the dependence of the hydrolysis level of the substrate - ABZ 1 -Asp 2 -Thr 3 -Phe 4 - Ile 5 -NH2, on pH conditions.
- This example presents the synthesis of one representative compound according to the present invention, namely the compound: ABZ 1 - Asp 2 -Thr 3 -Phe 4 -Ile 5 -NH2.
- the remaining peptides according to the invention can be synthesized in an analogous way.
- the superscripts indicate subsequent positions of residues in the compound according to the invention and the sequence of attachment of the residues during synthesis.
- the compounds according to the invention can be alternatively represented by an analogous formula without the indication of residue positions, which does not change the sequence of residues in the compounds according to the invention, as it remains unchanged. 1.
- the first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis, on a solid support, using Fmoc/tBu chemistry, i.e. with the use of protection.
- Boc-ABZ Fmoc-Asp(OtBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Ile.
- the synthesis of the compound according to the invention which can be used as a diagnostic marker for the detection of ovarian cancer, was carried out on a solid support enabling the conversion of 5-amino-2-beznoic acid into ANB-NH2 amide, namely amide resin TentaGel S RAM from RAPP Polymere (Germany), with a deposition of 0.23 mmol/g.
- amide resin TentaGel S RAM from RAPP Polymere (Germany)
- any other amide resin e.g. Rink Amide (Germany).
- All the obtained final compounds contained 2-aminobenzoic acid (ABZ) at the position 1 of their sequence, i.e. at the N-terminus, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at the position 6, i.e. at the C-terminus.
- ABZ acts here as a fluorescence donor
- ANB- 5- amino-2-nitrobenzoic acid - acts as a fluorescence quencher and simultaneously a chromophore.
- the peptides contained at least and preferably one reactive site in their sequence, located between the amino acid residue Ile-ANB-NTL, i.e. at the position 5 of the compound.
- the synthesis consisting in attaching amino acid derivatives is carried out from residue 6 to 1, i.e. from the C- to N-terminus.
- the synthesis of peptides was performed on TentaGel S RAM resin from Rapp Polymere with a deposition of 0.23 mmol/g.
- the resin was prepared, including loosening it by the wash cycle.
- the protection of the Fmoc amino group was removed from the solid support with the 20% solution of piperidine in NMP.
- the solvent washing cycle was carried out.
- a chloranil test was performed.
- the chloranil test consisted in transferring, by means of a spatula, several grains of resin from the reactor - a syringe, into a glass ampule, to which subsequently 100 pL of saturated solution of p-chloranil in toluene and 50 pL of fresh acetaldehyde were added. After 10 minutes, the control of grains colour was carried out.
- the first step in the synthesis of the peptide was deposition of ANB on 1 g of resin.
- the resin used for the reaction was washed with the following solvents: DMF, DCM and again DMF, after which the Fmoc- protection was removed from the functional group of the solid support.
- One cycle of removing the Fmoc- protection comprised the following steps:
- the resin with a free amino group was washed with 5% solution of A-methylmorpholine (NMM) in DMF, and then DMF.
- NMM A-methylmorpholine
- DMF diisopropylethylamine
- the corresponding amino acid derivative (9-fold molar excess relative to resin deposition) was dissolved in pyridine and was transferred to the flask containing the resin with deposited ANB. The whole was cooled until the temperature of -15°C was reached (ice bath: 1 part by weight of NH4CI, 1 part by weight of NaNO,, 1 part by weight of ice). After the desired temperate was reached, POCh was added (in 1 : 1 ratio to the amount of amino acid derivative used) and the whole was stirred on a magnetic stirrer: for 20 minutes at -15°C, 30 minutes at room temperature and 6 hours at 40°C (oil bath). When the reaction was completed, the resin was filtered off under reduced pressure, washed with DMF and MeOH and left to dry. In the next stage, the residue was attached in P2 position (Fmoc-Phe(tBu)).
- the amide of ABZ-Asp-Thr-Phe-Ile-ANB-NH2 peptide was removed from the solid support and simultaneously the side protection was removed using the mixture: TF A: phenol: water: TIPS (88:5:5:2, v/v/v/v) in a round-bottom flask on a magnetic stirrer.
- the identity/characteristics of the novel compound according to the invention were confirmed using the HPLC analysis.
- the conditions of the HPLC analysis were as follows: RP Bio Wide Pore Supelco C8 column, 250 mm 4 mm, a phase system A 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL/min, UV detection at 226 nm.
- Example 2 Testing the properties of the compound according to the invention as a cancer marker
- the activity of the novel compounds according to the invention was studied in a group of 20 subjects diagnosed with ovarian cancer using the representative compound according to the invention.
- the mechanism of action of the compounds according to the invention, including the representative compound having formula 2 consists in specific enzymatic cleavage, more specifically enzymatic hydrolysis, at the position that leads to the release of free molecules of respective chromophores: ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid) in the case of the compound having formula 2 or pNA (para-nitroanilide) in the case of the compound having formula 3, which exhibit absorbance at a wavelength of 320-480 nm, especially 380-430 nm, in particular 405 nm.
- the remaining compounds according to the invention are characterized by the analogous mechanism of action.
- the representative compound according to the invention ABZ 1 - Asp 2 -Thr 3 -Phe 4 -Ile 5 -NH2
- the measurement was performed on a 96-well plate designed for measuring absorbance and each sample was analysed in triplicate at the temperature of 37 °C. The duration of the measurement was 60 minutes.
- the wavelength characteristic for the chromophore (ANB-NH2) being released was monitored at the wavelength 405 nm (range 380-430 nm).
- the RP HPLC analysis of a randomly selected system comprising urine taken from a person diagnosed with ovarian cancer indicates that the compound according to the invention cleaves into the peptide fragment ABZ-Asp-Thr-Phe-Ile-OH and the chromophore group of the compound (ANB-NH2).
- the measurement showed that the colour intensity of the solution increased with time in all urine samples taken from persons diagnosed with ovarian cancer.
- the observed magnitude of increase in absorbance in time is different for each of the examined samples. A different effect was obtained for 20 samples from healthy subjects since no increase in absorbance within the diagnostic range was observed in any of the 20 tested urine samples.
- the results of the performed tests are presented in Fig. 3 and they indicate that the tested substrate, i.e. ABZ 1 - Asp 2 -Thr 3 -Phe 4 -Ile 5 -NH2, incubated with the samples taken from patients with diagnosed following cancers: intestine cancer, kidney cancer, prostate cancer, pancreas cancer, stomach cancer, lung cancer, uterine body cancer, and liver cancer, is not subject to cleavage and does not cause an increase in absorbance within the specified range.
- the samples tested were, in each case, a mixture of 20 samples derived from each of the cancers studied. This indicates cleavage selectivity of the compounds according to the invention, which makes them suitable for the specific detection of enzymatic activity specific to ovarian cancer and specific diagnosis of ovarian cancer.
- the analyses carried out confirmed suitability of the compounds according to the invention for the sensitive and specific detection of enzymatic activity specific to ovarian cancer and, by the same, their suitability also for the specific diagnosis of ovarian cancer, and as a diagnostic marker for ovarian cancer.
- the mechanism of action of the compounds according to the invention consists in their specific enzymatic cleavage at the position that leads to the release of free chromophore molecules, which generates a measurable optical signal that can be used for diagnostic purposes, in particular in the diagnosis of ovarian cancer according to the present invention.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a novel chemical compound - a diagnostic marker for use in medicine, more specifically in cancer diagnosis, in particular the diagnosis of ovarian cancer. The invention also relates to an in vitro method for detecting enzymatic activity present in a subject's body fluid, in particular derived from ovarian cancer cells, using the compound. The invention further relates to an in vitro method for diagnosing ovarian cancer using the compound, a kit comprising the compound and use of the compound for the detection of enzymatic activity specific to ovarian cancer and use of the compound for the diagnosis of ovarian cancer. The invention also relates to the compound for use as a diagnostic marker of ovarian cancer and a method for the treatment of ovarian cancer comprising a step of carrying out the method for the diagnosis of ovarian cancer as defined above using the compound.
Description
Compound - diagnostic marker for ovarian cancer, method for detecting enzymatic activity, method for diagnosis of ovarian cancer, kit comprising the compound, uses of the compound and method for the treatment of ovarian cancer
The invention relates to a new chemical compound, a diagnostic marker, for use in medicine, more specifically in cancer diagnostics, in particular diagnosis of ovarian cancer. The invention also relates to an m vitro method for detecting enzymatic activity present in a subject’s body fluid, in particular deriving from ovarian cancer cells, using the compound, an in vitro method for the diagnosis of ovarian cancer using the compound, a kit comprising the compound, use of the compound for the detection of enzymatic activity specific to ovarian cancer, use of the compound for the diagnosis of ovarian cancer, the compound for use as a diagnostic marker for ovarian cancer. The invention further relates to a method for the treatment of ovarian cancer comprising a step of carrying out the method for the diagnosis of ovarian cancer as specified above.
Background of the invention
Ovarian cancer is the 8th most common cancer diagnosed in women and the third cancer of the female reproductive organs. More than 300 000 new cases were reported in 2020. More than 114 000 women with ovarian cancer die each year worldwide. Risk factors for this cancer include childlessness, early or late menstruation, late menopause (after the age of 55), hormone therapy and active or passive smoking. In its early stages, ovarian cancer usually does not produce any symptoms; it is only when the disease is more advanced that gastrointestinal symptoms appear. Despite its name, ovarian cancer is not only a disease of the ovaries themselves - in most cases, so-called peritoneal spread is observed, i.e. the presence of tumours on other abdominal organs. Poor treatment results are due to late diagnosis and the fact that the cancer spreads very early throughout the abdomen, spreading to the intestines, liver, spleen and other tissues. This is due to the fact that the ovaries are organs that lie freely in the abdominal cavity, and therefore the cancer cells have great ease in spreading to neighbouring organs. In addition, it very often gives metastases via the lymphatic route to the lymph nodes. Ovarian cancer is characterised by moderate to poor prognosis, which depends primarily on the stage of the disease, as well as the age and general health of the patient. The earlier this cancer is detected, the greater the chance of a permanent cure. The 5-year survival rate for early detected cases exceeds 90%, while in advanced stages it is only 20%.
Symptoms of ovarian cancer are not specific and are usually of a general nature such as ascites, lower abdominal pain, bleeding from the reproductive tract. The diagnosis is confirmed by the result of the patient's gynaecological examination, ultrasound examination and the CA-125 marker present in the blood. Ca-125 is useful in the diagnosis, monitoring and prognosis of the course of the disease and the recognition of its recurrence. In about half of patients with early-stage ovarian cancer, it manifests itself with an elevated CA-125 level. It is estimated that more than 80 per cent of women with ovarian cancer have an elevated CA-125 marker level. The sensitivity and specificity of CA-125 in predicting the occurrence of malignant tumours, as well as its positive predictive value, are low, since before surgery, CA-125 levels are normal in about 50 per cent of patients with stage I epithelial ovarian cancer. In addition, increased CA-125 levels are influenced by a number of conditions, including the presence of malignancies of other abdominal organs and many benign conditions. Attempts are being made to use CA-125 as a predictive marker, but the use of this marker for diagnostic tests for the detection, including early, ovarian cancer is not recommended due to the low selectivity of this test.
A new qualitative serum test to help predict the diagnosis of ovarian cancer in women with pelvic tumours has been released for sale. This test, known as OVA1, is based on the assessment of five biological markers: transthyretin, apolipoprotein A-l, microglobulin P2, transferrin and CA-125.
Another approved biological marker is the determination of HE4 (human epididymis protein 4 from epithelial cells, human epididymis protein 4). HE4 is a protease inhibitor found in malignant epithelial ovarian cancer cells. It can be detected in the serum of women with ovarian cancer. The combined determination of HE4 and CA-125 has diagnostic parameters superior to both results interpreted alone. Based on a model that relies on the simultaneous determination in serum of two circulating cancer markers: CA-125 and HE4, the ROMA (Risk of ovarian malignancy algorithm), an algorithm for assessing the risk of the presence of epithelial ovarian cancer and the likelihood that an existing small pelvic lesion of unclear status is malignant, was created. Despite advances, there is no laboratory test, including a cancer marker, or set of tests that can diagnose ovarian cancer early and with certainty. Basis of the invention action
It is known that the process of initiation, growth and dissemination of cancer cells involves many factors, including many enzymes, in particular hydrolytic enzymes, especially proteolytic enzymes. Such enzymes catalyse enzymatic cleavage (hydrolytic or proteolytic)
of proteins and peptides into smaller fragments thereof. This process enables cancer cells to expand by colonizing new tissues, enhancing the process of blood vessels formation (angiogenesis), which enables effective delivery of nutrients to a tumour. Moreover, these enzymes are present as a result of death of healthy cells due to a tumour growth process. All these processes form a characteristic and specific profile of the enzymatic (proteolytic) activity of cancer cells, characteristic to a tumour.
In this field, there are known chromogenic peptide molecules that undergo enzymatic breakdown into smaller fragments resulting in a change or increase in the colour of the solution being tested. This chromogenic effect is a consequence of the release of a chromophore (e.g. 4-nitroanilide or 2-aminobenzoic acid) from a chromogenic peptide molecule.
This type of chromogenic molecules and their uses are known, for example, from the publication by Erlanger BF, Kokowsky N, Cohen W., “The preparation and properties of two new chromogenic substrates of trypsin”, Arch Biochem Biophys., November 1961; 95:271-8 and Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K. Amino acids and peptides. XXXV. “Facile preparation of p-nitroanilide analogs by the solid-phase method”, Chem Pharm Bull (Tokyo), November 2000; 48(11): 1740-4.
However, the use of this class of compounds in the diagnosis of ovarian cancer has not been described so far.
Methods for obtaining chromogenic peptides which consist in attaching individual components in appropriate time and stoichiometric conditions are also known in the prior art. The process of attaching consists of subsequent steps in which individual elements (amino acid derivatives) are attached, residues are washed off and protecting groups are sequentially removed and washed again. This cycle is repeated for each amino acid residue. The obtained peptide is separated from resin by a reaction in acidic conditions. Subsequently, the solution is separated from resin in the filtration process and then the peptide is precipitated from the solution by means of a non-polar solvent.
Chromogenic peptide compounds appropriate for a specific and early diagnosis of ovarian cancer or methods to obtain them are however not known in the prior art.
Therefore, in this field there is an urgent need for “a cancer marker” for ovarian cancer, which would enable an early, sensitive and specific diagnosis of ovarian cancer in a non- invasive and reliable manner, and for diagnostic methods and treatment methods using such a diagnostic marker.
The object of the present invention is to provide a novel, specific diagnostic marker for ovarian cancer and diagnostic methods using such a marker for a non-invasive, quick, sensitive and specific, early detection of ovarian cancer, which would also be appropriate for screening tests, as well as treatment methods using such a marker.
These objects have been achieved by the inventions defined in the attached patent claims, whereas preferred variants thereof are defined in the dependent claims.
Summary of the invention
The invention provides a compound having formula 1 :
Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1), wherein XI comprises or consists of molecule Cl, and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of florescence donor and fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into the fragments Xl-Asp-Thr- Phe-Ile-OH (Fragment 1) and X2 (Fragment 2) with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
The compound according to the invention preferably undergoes hydrolytic cleavage, more preferably proteolytic.
Preferably, in the compound according to the invention the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ)/ 5-amino-2-nitrobenzoic (ANB), (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/TyrQ-NCh), more preferably the pair of Cl and C2 is (ABZ)/pNA or ABZ/ANB-NH2.
Preferably, the compound according to the invention is a compound having formula 2: ABZ- Asp-Thr-Phe-Ile-ANB-NFF (formula 2) or a compound having formula 3: ABZ-Asp-Thr- Phe-Ile-pNA (formula 3).
More preferably, the compound according to the invention undergoes hydrolytic cleavage with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2.
The invention further provides an in vitro method for detecting enzymatic activity present in a subject’s body fluid, in particular deriving from ovarian cancer cells, comprising: a) contacting a sample of body fluid with the compound having formula 1 : Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1),
wherein XI comprises or consists of molecule Cl and X2 comprises or consist of molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the said compound undergoes enzymatic cleavage into the fragments Xl-Asp- Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), and b) detecting a measurable optical signal which is generated upon spatial separation of molecules Cl and C2.
In the method for detecting enzymatic activity according to the invention, enzymatic activity is preferably hydrolytic activity, more preferably proteolytic activity.
In the method for detecting enzymatic activity according to the invention as the said compound the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NHz (formula 2) or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is preferably used.
In the method for detecting enzymatic activity according to the invention as the said body fluid preferably a urine, more preferably human urine, is used.
The invention also relates to an in vitro method for the diagnosis of ovarian cancer, wherein the presence or absence of ovarian cancer in a subject is detected by measuring enzymatic activity specific to ovarian cancer in a body fluid sample from an examined subject, and wherein the absence of the said enzymatic activity indicates the absence of ovarian cancer, whereas the presence of the said enzymatic activity indicates the presence of ovarian cancer. In the method for detecting/diagnosis of ovarian cancer according to the invention, the detection of enzymatic activity is carried out by the method for detecting enzymatic activity as defined above.
In the method for detecting/diagnosis of ovarian cancer according to the invention, the measurement of the said enzymatic activity is performed using the compound having formula 1 :
Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1), wherein XI comprises or consists of molecule Cl and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor,
and wherein the said compound undergoes enzymatic cleavage into the fragments Xl-Asp- Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) with the generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
In the method for detecting/diagnosis of ovarian cancer according to the invention, the said body fluid sample is preferably incubated with the said compound in a measurement buffer having neutral or alkaline pH, more preferably physiological, within the range of sample-to- measurement buffer ratio of from 1 :2 to 1 : 10, preferably 1 :5.
In the method for detecting/diagnosis of ovarian cancer according to the invention, the said compound is preferably used at a concentration of 0.1-10 mg/mL, in particular 0.25-7.5 mg/mL.
In the method for detecting/diagnosis of ovarian cancer according to the invention, as the said compound the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (formula 2) or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is preferably used.
In the method for detecting/diagnosis of ovarian cancer according to the invention, as the said sample a urine sample, more preferably human urine, is preferably used.
In the method for detecting/diagnosis of ovarian cancer according to the invention, the measurement of the said enzymatic activity preferably comprises the measurement of absorbance intensity within the range of 300-500 nm, more preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, more preferably 36-38° C.
The invention further provides a kit comprising any compound according to the invention as defined above and a measurement buffer.
In the kit according to the invention, the said compound is preferably the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp- Thr-Phe-Ile-pNA.
The invention also provides the use of any compound according to the invention as defined above for the detection of enzymatic activity specific to ovarian cancer.
The invention also provides the use of any compound according to the invention as defined above for the diagnosis of ovarian cancer.
Preferably, in such use the diagnosis of ovarian cancer comprises the detection of primary ovarian cancer, detection of Minimal Residual Disease after surgical resection of ovarian cancer and/or detection of ovarian cancer recurrence.
Preferably, the compound in the uses according to the invention is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NJL or a compound having formula 3: ABZ-Asp- Thr-Phe-Ile-pNA.
The invention further provides any of the compounds according to the invention as defined above for use as a diagnostic marker for the detection of ovarian cancer.
Preferably, the compound for use as the diagnostic marker according to the invention is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFF or a compound having formula 3 : ABZ-Asp-Thr-Phe-Ile-pNA.
The invention further provides a method for the treatment of ovarian cancer, wherein a) the presence of enzymatic activity specific to ovarian cancer is detected by any method as defined above in a body fluid sample from an examined subject, and b) if the presence of the said enzymatic activity is found in the said sample, a treatment for ovarian cancer is applied in the subject.
Preferably, in the method for the treatment according to the invention, after the end of the treatment in accordance with point b), the said enzymatic activity specific to ovarian cancer is monitored at predetermined time intervals.
Preferably, in the method for the treatment according to the invention, a urine sample, preferably human urine, is used as the sample.
Preferably, in the method for the treatment according to the invention, the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFF or a compound having formula 3: ABZ-Asp- Thr-Phe-Ile-pNAis used as the said compound.
Detailed description of the invention
It is to be understood that the present invention is defined in the appended claims. The present description illustrates various, non-limiting embodiments and examples of the invention. The present invention is not limited to any particular methodology, protocol or reagents used to carry it out, unless indicated otherwise. The terms as well as scientific and technical expressions as used herein have meanings commonly known and used by persons skilled in the art of the present invention. For the sake of clarity however, the following expressions/terms and acronyms used in the patent shall be understood as follows:
A chromogenic compound or a chromogenic molecule means a compound having chromogenic properties. Chromogenic properties mean the ability of a compound to form a coloured product.
A fluorescent compound or a fluorescent molecule means a compound having fluorogenic properties. Fluorogenic properties mean the ability of a compound to form a product emitting fluorescence.
NMP stands for A-methylpirrolidone; DMF stands for dimethylformamide; DCM stands for methylene chloride or dichloromethane; pNA stands for 4-nitroaniline or para-nitroaniline; ABZ stands for 2-aminobenzoic acid, ANB-NH2 stands for amide of 5-amino-2-nitrobenzoic acid; Boc stands for tert-butyloxycarbonyl group; Fmoc stands for 9- fluorenylometoxycarbonyl group; and TFA stands for trifluoroacetic acid.
In the context of this invention, the term ovarian cancer shall be understood to mean primary cancer of ovary (malignant neoplasm) that develops from tissues located in the ovary. Ovarian cancer is most frequently endometrial adenocarcinoma (about 90 %), less frequently serous clear cell cancer. The term: ovarian cancer as used herein comprises thus all malignant ovarian neoplasms which develop from the tissues located in the ovary.
In the context of the present invention, the term diagnosis of ovarian cancer shall be understood to mean identification of this disease, in particular at its early stage at which other diagnostic methods are not sensitive and/or specific enough. As used herein, the diagnosis of ovarian cancer also comprises the detection of Minimal Residual Disease (MRD) after surgical resection of ovarian cancer and detection of ovarian cancer recurrence after previously completed treatment of ovarian cancer.
In the context of the present invention, the term: treatment of ovarian cancer shall be understood to mean a treatment at an early stage of progression of the disease, which makes it possible to significantly prolong survival time and improve the quality of life of the diseased subjects.
In the context of the present invention, the term: monitoring shall be understood to mean the diagnosing of Minimal Residual Disease (MRD)) - the presence of a small number of cancer cells that have survived in the organism (during treatment or remission), in the amounts undetectable by means of standard diagnostic methods.
In the context of the present invention, the term: subject shall be understood to mean a human subject or a mammal that is suspected to have ovarian cancer, or alternatively a human subject or a mammal belonging to a group with an increased risk of ovarian cancer, or a human subject or a mammal after resection of ovarian cancer or after a finished treatment of ovarian cancer. The subject is preferably a human subject.
The compounds according to the invention have chromogenic properties due to the presence of a chromophore, and fluorogenic properties, i.e. they contain molecules of a fluorescence donor and acceptor. Due to their structure, developed in such a way that an increase in colour is observed in the wavelength range of 380-440 nm, specifically as a result of contact of a tested body fluid sample from an examined subject with ovarian cancer, whereas such an effect is not observed in the reaction with a body fluid sample from a healthy subject or a subject with the diagnosis of another type of cancer, these compounds make it possible to detect enzymatic activity specific to ovarian cancer, and in particular to diagnose ovarian cancer in a specific and sensitive manner, also at an early stage of progression of this cancer. The examined subject is preferably a human subject. The body fluid is preferably urine, more preferably human urine.
In the first aspect of the present invention, a novel chemical compound is provided which compound has formula 1 :
Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1), wherein XI is an amino acid derivative or a peptide fragment comprising molecule Cl, or XI consists of such a molecule Cl, and X2 is an amino acid derivative or a peptide fragment comprising molecule C2, or X2 consists of such a molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor. The superscripts indicate subsequent positions of residues in the compound according to the invention and the sequence of attaching of the residues during synthesis. In accordance with the present invention, in this context chemical formula 1 can be alternatively written without indicating the numbering of residues. The core of all compounds according to the invention is tetrapeptide with the indicated sequence of 4 amino acids, i.e. Asp-Thr-Phe-Ile (the notation in the three-letter amino acid abbreviation format equivalent to the notation: DTFI in the one-letter amino acid abbreviation format), which sequence is also presented in the Sequence Listing as sequence no. 1.
The compound according to the invention undergoes enzymatic cleavage into the fragments: Xl-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) with the generation of a measurable optical signal upon spatial separation of molecules Cl and C2. The measurable optical signal is measured by a method for measuring a change in absorbance/fluorescence after the enzymatic cleavage of the compound. Preferably, molecules Cl and C2 are separated from each other by not more than 10 amino acid residues, which ensure efficient quenching of the fluorescence donor by the fluorescence acceptor. It is obvious for the
skilled person that the key factor is the distance between the fluorescence donor and acceptor. Therefore, where the amino acid sequence separating molecules Cl and C2 is folded into a twisted or condensed secondary structure, resulting in a proximity of molecules Cl and C2 relative to the primary structure, the distance between molecules Cl and C2 can be greater than 10 amino acid residues.
The compound, due to its chromogenic properties and the presence of a reactive site at the position 5 enabling enzymatic (preferably proteolytic) cleavage into smaller fragments, is particularly suitable for use as a diagnostic marker, in particular a specific diagnostic biomarker for ovarian cancer, in particular for use in the early diagnosis of ovarian cancer. In preferred embodiments, the compound according to the invention undergoes hydrolytic cleavage, more preferably proteolytic cleavage.
In preferred embodiments the pair of molecules Cl and C2 is selected from a group consisting of: 2-aminobenzoic acid (ABZ)/5-amino-2-nitrobenzoic acid (ANB), (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO2), more preferably the pair of molecules Cl and C2 is ABZ/pNA or ABZ/ANB-NH2.
In preferred embodiments, the compound according to the invention is: the compound having formula 2:
ABZ1-Asp2-Thr3-Phe4-Ile5-ANB6-NH2 (formula 2) or the compound having formula 3 :
ABZ1-Asp2-Thr3-Phe4-Ile5-pNA6 (formula 3), wherein ABZ stands for 2-aminobenzoic acid, ANB-NH2 stands for amide of 5-amino-2- nitrobenzoic acid, and pNA stands for 4-nitroaniline.
The compound is subject to hydrolytic cleavage with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2 in the case of the compound having formula 2, whereas in the case of the compound having formula 3, with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: pNA. Thus, fragments 2 are free chromophores.
Spatial separation of molecules Cl and C2 being a result of the enzymatic cleavage of the compound according to the invention causes generation of a measurable optical signal because fluorescence emitted from the fluorescence donor is no longer quenched by the fluorescence acceptor. Such measurable optical signal can be detected preferably at a wavelength of 300-500 nm, more preferably 380-430 nm.
The compounds according to the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides which consists in carrying out the process on a solid support in the form of a resin having an Fmoc group, which is removed in the course of the reaction. For example, it can be an amide resin, e.g. Teenage S RAM or RinkAmide, but any other commercially available resin can also be used. The resin used to carry out the process should be properly prepared. The preparation of the resin consists in increasing its volume by repeated washing with hydrophobic solvents. Preferably, a resin with a deposition of 0.23 mmol/g is used. The Fmoc protecting group must be removed from the resin by washing it with a 20% solvent solution.
Then, the known processes for obtaining chromogenic peptides comprise attaching individual components in appropriate time and stoichiometric conditions. The attaching process consists of subsequent steps in which individual elements (amino acid derivatives) are attached, residuals are washed off and protecting groups are successively removed and washed again. This cycle is repeated for each amino acid residue. The obtained peptide is separated from the resin by a reaction under acidic conditions. Then, the solution is separated from the resin in the filtration process and then the peptide is precipitated from the obtained solution by means of a non-polar solvent. The peptide precipitate obtained in this way is centrifuged.
Exemplary, detailed but non-limiting, method for the synthesis of the compounds according to the invention is described below and in Example 1 below.
The method for the synthesis of the compound according to the invention consists in that the process is carried out on a solid support in the form of a resin, preferably having an Fmoc group, wherein before the start of the process the solid support is prepared by increasing its volume by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removing the Fmoc protecting group, preferably by washing with 10 - 30 % piperidine solution, in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone.
Then, the method is carried out in subsequent steps: a) Deposition of 5-amino-2 -nitrobenzoic acid, ANB (or another chromophore suitable for use according to the invention as defined in the claims) on the resin is preceded by washing the solid support with a 3-6% solution of N-m ethylmorpholine (NMM) in DMF, and then DMF, after which a solution of ANB in DMF is prepared to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are successively added in the following excess
relative to the polymer deposition: ANB/TBTU/DMAP/DIPEA, 3:3:2:6; the mixture prepared in this way is added to the resin and is mixed until homogenous, after which the resin is filtered off under reduced pressure and is washed with solvents such as DMF, DCM and isopropanol, and then the attaching of ANB to the resin is continued using hexafluorophosphate-O-(7-azabenzotriazol-l-yl)-A,A,A’,7V’-tetramethyluronium (HATU), and then hexafl uorophosphate-(9-(benzotri azol - 1 -yl)-A, N, N ’N ’-tetramethyluronium
(HBTU) in excess, and after finishing, the solid support is washed successively with DMF, DCM and isopropanol, and is gently dried; b) Attachment of the amino acid residue to ANB is carried out by a reaction with the amino acid derivative - Fmoc-Ile-OH, wherein at least fivefold molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and is contacted with the resin with deposited ANB, after which the whole is cooled to a temperature not lower than -20°C, and then POCh is added in the ratio 1 : 1 relative to the amount of the amino acid derivative used and the whole is mixed, after which the mixing process is carried out at room temperature and then at an elevated temperature and when the reaction is completed, the resin in filtered off under reduced pressure, washed with DMF and MeOH and gently dried, after which the obtained intermediate compound is subjected to the process of acylation, successively attaching a fragment of Asp-Thr-Phe; c) Acylation of the obtained intermediate compound is carried out using an amino acid derivative, preferably Fmoc-Phe-OH, and subsequently Fmoc-Thr(tBu)-OH, then Fmoc- Asp(OtBu)-OH and, at the final step of the synthesis, Boc-Abz-OH, the acylation being carried out in steps from residue 6 to 1, using diisopropylcarbodiimide as a coupling agent, which is used in excess, and after finishing each step the resin is washed with DMF and preferably is subjected to the chloranil test (a test for the presence of free amino groups) in which the attachment of the amino acid derivative is monitored; d) Removal of the Fmoc protecting group is carried out by washing with 10 - 30% piperidine solution in DMF and subsequent washings with each of the solvents: DMF, isopropanol and methylene chloride; e) Separation of the peptide from the resin is carried out using the mixture: TFA:phenol:water:TIPS while maintaining the ratios of 88:5:5:2 N/N/N/N, respectively, the mixture is stirred for the period of at least one hour, preferably three hours, and the obtained precipitate filtered off under reduced pressure, after which it is washed with diethyl ether and the obtained peptide is centrifuged;
f) Preparation of the finished product is carried out by dissolving the peptide in water by means of ultrasound and then it is subjected to lyophilisation.
The second aspect of the present invention provides an n vitro method for detecting enzymatic, preferably proteolytic, activity, present in a subject’s body fluid, in particular deriving from ovarian cancer cells, the method comprising a) contacting the body fluid sample with the compound according to the invention and b) detecting a measurable optical signal which is generated upon spatial separation of molecules Cl and C2 present in the compound according to the invention. In a preferred embodiment of this aspect, the examined subject in this case is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, in particular human urine.
In the preferred embodiment of this aspect, the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NHz or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA is used.
The third aspect of the present invention provides an in vitro method for the diagnosis of ovarian cancer in which the presence or absence or ovarian cancer in a subject is detected by measuring enzymatic activity specific to ovarian cancer in a body fluid sample from the examined subject, wherein the absence of the said enzymatic activity indicates the absence of ovarian cancer whereas the presence of the said enzymatic activity indicates the presence of ovarian cancer. Such detection of enzymatic activity is preferably carried out using methods for detecting enzymatic activity as discussed above. In the preferred embodiment of this aspect the subject is a human subject. In the preferred embodiment of this aspect the body fluid is urine, in particular human urine. In the preferred embodiment of this aspect the enzymatic activity specific to ovarian cancer is proteolytic activity. In the preferred embodiment of this aspect the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA is used.
Moreover, in the preferred embodiment of this aspect, the measurement of the said enzymatic activity in the methods according to the invention comprises the measurement of absorbance intensity within the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, preferably 36- 38° C. This enables obtaining a maximally intensive measurable optical signal resulting from an increase in absorbance or fluorescence.
Furthermore, in the preferred embodiments of the said methods according to the invention the measurement of the said enzymatic activity is performed using the compound according to the invention in the range of concentrations of 0.1 - 10 mg/mL, more preferably at the concentration of 1 mg/mL. In the preferred embodiments of the said methods according to the invention the tested sample is incubated with the compound according to the invention in a measurement buffer having a neutral or alkaline pH, preferably physiological, with a body fluid sample, preferably human urine, with the sample (e.g. of urine) to measurement buffer ratio ranging from 1 :2 to 1 :10, preferably 1 :5. The sample is preferably taken from a subject with a referral for the diagnosis of ovarian cancer. Preferably, absorbance intensity is measured within the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, preferably 36-38° C. In the aforementioned conditions, a maximally intensive measurable optical signal is obtained resulting from an increase in absorbance or fluorescence.
In the fourth aspect, the present invention provides a kit comprising any compound according to the invention and a measurement buffer. Measurement buffers are known in this art and a buffer suitable for use in the kit according to the invention is, for example, but without limitation, the Tris-HCl buffer. In the preferred embodiment, in the kit according to the invention the said compound is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.
In the fifth aspect, the present invention provides use of the compound according to the invention for the detection of enzymatic activity specific to ovarian cancer. In the sixth aspect, the present invention provides use of the compound according to the invention for the diagnosis of ovarian cancer. Preferably, the diagnosis of ovarian cancer comprises, according to the invention, the detection of primary ovarian cancer, detection of Minimal Residual Disease after surgical resection of ovarian cancer and/or detection of ovarian cancer recurrence after previously completed ovarian cancer treatment.
In the seventh aspect, the present invention provides the compound according to the invention for use as a diagnostic marker for the detection of ovarian cancer. In the preferred embodiments of this aspect the said compound is the compound having formula 2: ABZ- Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA. In the eight aspect, the present invention provides a method for the treatment of ovarian cancer wherein
a) the presence of enzymatic activity specific to ovarian cancer is detected by any method according to the invention as defined above, in a body fluid sample from the examined subject, and b) when the said enzymatic activity is found to be present in the said sample, a treatment of ovarian cancer is applied in the subject.
In the preferred embodiment of the method for the treatment, after completing the treatment according to point b), the said enzymatic activity specific to ovarian cancer is monitored at predetermined time intervals as known in the art, e.g. every week, every several weeks, every month, every several months, every year or at any other intervals considered to be appropriate by the skilled person, in order to detect Minimal Residual Disease after surgical resection of ovarian cancer or recurrence. Furthermore, in the preferred embodiment of the method, a urine sample, preferably human urine, is used as the test sample. In the preferred embodiment of the method for the treatment the compound having formula 2: ABZ-Asp- Thr-Phe-Ile-ANB-NHz (formula 2) or a compound having formula 3 : ABZ-Asp-Thr-Phe- Ile-pNA (formula 3) is used as the said compound.
The advantages of the present invention consist in providing a novel chemical compound having properties that make it suitable for use for specific and sensitive detection of enzymatic activity specific to ovarian cancer, for use as a diagnostic biomarker for the detection of ovarian cancer, for use in a fast, non-invasive diagnosis of ovarian cancer, while enabling the detection of ovarian cancer at an early stage of its progression. Another advantage is that the diagnostic methods according to the invention can be successfully used in screening tests. This enables full diagnosis at an early stage of cancer progression and consequently a more effective treatment. Early diagnosis enables surgical treatment, which significantly prolongs patient’s survival time. It is also important when monitoring the effectiveness of the applied surgical treatment and/or chemotherapy of ovarian cancer since it is possible to detect Minimal Residual Disease or recurrence, if any.
The present invention will now be illustrated in the following figures and the following examples, which however are not intended to limit, in any manner, the scope of the invention as defined in the claims.
Brief description of the figures
Fig. 1 shows the results of chromatographic analysis of the substrate cleavage, i.e. ABZ-Asp-Thr-Phe-Ile-ANB-NFE, in a sample of urine from a subject with ovarian cancer.
Fig. 2 shows the rate of hydrolysis of the substrate - ABZ-Asp-Thr-Phe-Ile-ANB-NTL - in the samples of urine from subjects with diagnosed ovarian cancer (samples 1-20) and urine taken from healthy subjects (samples Z21 - Z40). Arabic numerals indicate the number of the selected urine sample.
Fig. 3 shows the selectivity of hydrolysis of the substrate - ABZ1-Asp2-Thr3-Phe4-Ile5- ANB6-NH2 (i.e. compound of formula 2) in urine samples taken from subjects with diagnosed ovarian cancer (sample 1) and urine samples taken from subjects with the diagnosis of another neoplastic disease (samples 2 - 9). Arabic numerals indicate the numbers of given cancer types. The samples tested for each type of cancer were derived from 20 different patients for each of the tested cancers. The results are mean values for given cancer types. The results show selectivity of substrate cleavage in the case of urine from patients with ovarian cancer as compared to urine samples from patients suffering from other neoplasms.
Fig. 4 shows the dependence of the hydrolysis level of the substrate - ABZ1-Asp2-Thr3-Phe4- Ile5-NH2, on pH conditions.
Examples
The invention is illustrated by the following non-limiting examples. Unless indicated otherwise, the examples below use known and/or commercially available devices, methods, reaction conditions, reactants and sets, which are commonly used in the field to which the present invention belongs and which are recommended by the producers of respective reactants and kits.
Example 1 : Synthesis of the compound according to the invention
This example presents the synthesis of one representative compound according to the present invention, namely the compound: ABZ1- Asp2-Thr3-Phe4-Ile5-NH2. The remaining peptides according to the invention can be synthesized in an analogous way. The superscripts indicate subsequent positions of residues in the compound according to the invention and the sequence of attachment of the residues during synthesis. The compounds according to the invention can be alternatively represented by an analogous formula without the indication of residue positions, which does not change the sequence of residues in the compounds according to the invention, as it remains unchanged.
1. Obtaining a chromogenic peptide a) The first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis, on a solid support, using Fmoc/tBu chemistry, i.e. with the use of protection.
A compound having the sequence ABZ1- Asp2-Thr3-Phe4-Ile5-NH2, wherein ABZ is 2- aminobenzoic acid and ANB-NH2 is amide of 5-amino-2-benzoic acid and ANB is 5-amino- 2-benzoic acid, was obtained in the process of solid phase chemical synthesis using the following amino acid derivatives:
Boc-ABZ, Fmoc-Asp(OtBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Ile.
The synthesis of the compound according to the invention, which can be used as a diagnostic marker for the detection of ovarian cancer, was carried out on a solid support enabling the conversion of 5-amino-2-beznoic acid into ANB-NH2 amide, namely amide resin TentaGel S RAM from RAPP Polymere (Germany), with a deposition of 0.23 mmol/g. However, it is possible to use any other amide resin, e.g. Rink Amide (Germany).
The synthesis of the compound was carried out manually using a laboratory shaker. In most of the steps a 25 mL sintered syringe for solid phase synthesis was used as a reactor.
All the obtained final compounds contained 2-aminobenzoic acid (ABZ) at the position 1 of their sequence, i.e. at the N-terminus, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at the position 6, i.e. at the C-terminus. ABZ acts here as a fluorescence donor, while ANB- 5- amino-2-nitrobenzoic acid - acts as a fluorescence quencher and simultaneously a chromophore. The peptides contained at least and preferably one reactive site in their sequence, located between the amino acid residue Ile-ANB-NTL, i.e. at the position 5 of the compound. The synthesis consisting in attaching amino acid derivatives is carried out from residue 6 to 1, i.e. from the C- to N-terminus. b) Deposition of ANB on TentaGel S RAM resin:
The synthesis of peptides was performed on TentaGel S RAM resin from Rapp Polymere with a deposition of 0.23 mmol/g. In the first step, the resin was prepared, including loosening it by the wash cycle. Subsequently, the protection of the Fmoc amino group was removed from the solid support with the 20% solution of piperidine in NMP. Then, the solvent washing cycle was carried out. In order to confirm the presence of free amino groups, a chloranil test was performed.
Solvent wash cycle:
DMF 1 x 10 minutes; IsOH 1 x 10 minutes; DCM 1 x 10 minutes.
Removal of Fmoc protection:
DMF 1 x 5 minutes; 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.
Solvent wash cycle:
DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes. c) Chloranil test:
The chloranil test consisted in transferring, by means of a spatula, several grains of resin from the reactor - a syringe, into a glass ampule, to which subsequently 100 pL of saturated solution of p-chloranil in toluene and 50 pL of fresh acetaldehyde were added. After 10 minutes, the control of grains colour was carried out.
At that stage, after performing the test, a green colour of the grains was obtained, which evidenced the presence of free amino groups. After confirming the removal of 9-fluorenylmethoxy carbonyl protection from the resin, it was possible to proceed to the next step, the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid). d) Deposition of 5-amino-2-nitrobenzoic acid on solid support:
The first step in the synthesis of the peptide was deposition of ANB on 1 g of resin. Before attaching the chromophore, the resin used for the reaction was washed with the following solvents: DMF, DCM and again DMF, after which the Fmoc- protection was removed from the functional group of the solid support. One cycle of removing the Fmoc- protection comprised the following steps:
Removal of Fmoc- protection:
20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes. e) Washing:
DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes. f) Chloranil test:
The resin with a free amino group was washed with 5% solution of A-methylmorpholine (NMM) in DMF, and then DMF. The procedure of removing the Fmoc- protection and the wash cycle were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, and TBTU, DMAP and finally diisopropylethylamine (DIPEA) were subsequently added in the following excess relative to polymer deposition: ANB/TBTU/DMAP/DIPEA, 3:3:2:6 N/N/N/N. The mixture prepared in this way was added to the resin and was stirred for 3 hours. The resin was filtered off under reduced pressure, washed with DMF, DCM and isopropanol, and the entire acylation procedure was repeated twice. To carry out subsequent
reactions of attaching ANB to the resin, hexafluorophosphate-O-(7-azabenzotriazol-l-yl)- N,N,N',N'-tetramethyluronium (HATU) and then hexafluorophosphate-( -(benzotriazol- l - yl)-N,N,N',N'-tetramethyluronium (HATU) were used. In the last step, the resin was washed successively with DMF, DCM and isopropanol, and was air dried. g) Attachment of the C-terminal amino acid residue (Fmoc-Ile-OH) to ANB:
The corresponding amino acid derivative (9-fold molar excess relative to resin deposition) was dissolved in pyridine and was transferred to the flask containing the resin with deposited ANB. The whole was cooled until the temperature of -15°C was reached (ice bath: 1 part by weight of NH4CI, 1 part by weight of NaNO,, 1 part by weight of ice). After the desired temperate was reached, POCh was added (in 1 : 1 ratio to the amount of amino acid derivative used) and the whole was stirred on a magnetic stirrer: for 20 minutes at -15°C, 30 minutes at room temperature and 6 hours at 40°C (oil bath). When the reaction was completed, the resin was filtered off under reduced pressure, washed with DMF and MeOH and left to dry. In the next stage, the residue was attached in P2 position (Fmoc-Phe(tBu)).
Every attachment of amino acid residue was preceded by washing the resin with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent attachments. The procedure was repeated twice.
After each acylation, a resin wash cycle was started and then the chloranil test was performed in order to monitor the attachment of the amino acid derivative to free amino acid groups of the resin.
Solvent wash cycle:
DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.
Chloranil test:
As a result of the performed tests, after the first two coupling procedures, the colour of the grains was first green and then grey, so it was necessary to perform another acylation, as a result of which the resin grains tested by the chloranil test were colourless. This evidenced the attachment of ANB to the TentaGel S RAM resin, and thus it was possible to proceed to the next step of peptide synthesis. h) Attachment of subsequent protected amino acid residues:
The resin together with the attached fragment ANB -He, located in the reactor, was washed with DMF, which was followed by deprotection of the Fmoc from the amino group in order to attach the protected amino acid derivative Phe.
Removal of Fmoc protection:
DMF 1 x 5 minutes; 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.
Solvent wash cycle:
DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.
Chloranil test:
The chloranil test produced a positive result, which was evidenced by the green colour of the resin grains. Thus, it was possible to proceed to the next step - attachment of the amino acid residue Fmoc-Thr(tBu)-OH.
Attachment of the amino acid derivative
The process of coupling was preceded by washing the resin with DMF. The composition of the coupling mixture remained unchanged when attaching the protected serine residue.
After the end of each acylation, a solvent wash cycle was performed according to the specified procedure and then the chloranil test for the presence of free amino acid groups in the solution was performed.
Solvent wash cycle
DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.
Chloranil test:
During the test performed after the second acylation the resin grains were colourless, and thus it was possible to proceed to the next step of the synthesis i.e. the introduction of another protected amino acid derivative - Fmoc- Asp(OtBu) and 2-aminobenzoic acid molecule. The coupling processes were performed according to the procedure discussed earlier.
Tests carried out after attaching the above-mentioned residues showed positive results - the resin grains were colourless.
2. Removal of the peptide from the solid support
After the synthesis, the amide of ABZ-Asp-Thr-Phe-Ile-ANB-NH2 peptide was removed from the solid support and simultaneously the side protection was removed using the mixture: TF A: phenol: water: TIPS (88:5:5:2, v/v/v/v) in a round-bottom flask on a magnetic stirrer.
After 3 hours, the content of the flask was filtered off under reduced pressure in sintered (Schott) funnels and washed with diethyl ether. The obtained sediment was centrifuged on a SIGMA 2K30 centrifuge (Laboratory Centrifuges) for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by means of ultrasound and then it was subjected
to lyophilisation. The remaining compounds according to the invention can be obtained in an analogous way.
The identity/characteristics of the novel compound according to the invention were confirmed using the HPLC analysis. The conditions of the HPLC analysis were as follows: RP Bio Wide Pore Supelco C8 column, 250 mm 4 mm, a phase system A 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL/min, UV detection at 226 nm.
The analysis carried out confirmed that the compound according to the invention was obtained.
Example 2: Testing the properties of the compound according to the invention as a cancer marker
The activity of the novel compounds according to the invention was studied in a group of 20 subjects diagnosed with ovarian cancer using the representative compound according to the invention. The mechanism of action of the compounds according to the invention, including the representative compound having formula 2, consists in specific enzymatic cleavage, more specifically enzymatic hydrolysis, at the position that leads to the release of free molecules of respective chromophores: ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid) in the case of the compound having formula 2 or pNA (para-nitroanilide) in the case of the compound having formula 3, which exhibit absorbance at a wavelength of 320-480 nm, especially 380-430 nm, in particular 405 nm. The remaining compounds according to the invention are characterized by the analogous mechanism of action. For this purpose, the representative compound according to the invention, ABZ1- Asp2-Thr3-Phe4-Ile5-NH2, was dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg/mL) and then 50 pL of the solution was mixed with 120 pL of a buffer (200 mM Tris-HCl, pH 8.0) and 80 pL of urine from a subject suffering from ovarian cancer. The measurement was performed on a 96-well plate designed for measuring absorbance and each sample was analysed in triplicate at the temperature of 37 °C. The duration of the measurement was 60 minutes. During the measurement, the wavelength characteristic for the chromophore (ANB-NH2) being released was monitored at the wavelength 405 nm (range 380-430 nm).
A shown in Fig. 1, the RP HPLC analysis of a randomly selected system comprising urine taken from a person diagnosed with ovarian cancer indicates that the compound according to the invention cleaves into the peptide fragment ABZ-Asp-Thr-Phe-Ile-OH and the chromophore group of the compound (ANB-NH2).
The measurement showed that the colour intensity of the solution increased with time in all urine samples taken from persons diagnosed with ovarian cancer. The observed magnitude of increase in absorbance in time is different for each of the examined samples. A different effect was obtained for 20 samples from healthy subjects since no increase in absorbance within the diagnostic range was observed in any of the 20 tested urine samples.
The tests carried out show that all samples 1-20 from persons suffering from ovarian cancer underwent cleavage, but in the case of samples 3 and 17, cleavage of the substrate, i.e. ABZ- Asp-Thr-Phe-Ile-ANB-NH2, proceeded less efficiently than in the case of samples 1 or 13 (Table 1 Fig. 2,). Such a result may be due to the difference in the activity as well as the amount of enzymes responsible for the enzymatic cleavage (proteolysis). Furthermore, the results presented in Table 1 below indicate that incubating the substrate solution - the compound according to the invention - with urine samples taken from healthy persons (without a cancer diagnosis, marked with Arabic numerals sequentially from Z21 to Z40) does not lead to an increase in absorbance and thus hydrolysis of the tested compound does not take place. The result indicates the absence of proteolytic enzymes specific/characteristic of ovarian cancer.
Table 1. Results of absorbance analysis
Furthermore, the dependence of the cleavage selectivity of the substrate, i.e. the compound according to the invention, on the cancer type being examined, was studied. The results of the performed tests are presented in Fig. 3 and they indicate that the tested substrate, i.e. ABZ1- Asp2-Thr3-Phe4-Ile5-NH2, incubated with the samples taken from patients with diagnosed following cancers: intestine cancer, kidney cancer, prostate cancer, pancreas cancer, stomach cancer, lung cancer, uterine body cancer, and liver cancer, is not subject to cleavage and does not cause an increase in absorbance within the specified range. The samples tested were, in each case, a mixture of 20 samples derived from each of the cancers
studied. This indicates cleavage selectivity of the compounds according to the invention, which makes them suitable for the specific detection of enzymatic activity specific to ovarian cancer and specific diagnosis of ovarian cancer.
Table 2 below presents the obtained measurements results for each sample in triplicate. Table 2. Results of analysis of selectivity of the cleavage
Furthermore, measurements concerning the dependence of the proteolytic activity of the representative compound according to the invention on the reaction pH were performed. The experiment has shown that the studied material has at least one enzyme exhibiting maximum activity at alkaline pH (Fig. 4).
The analyses carried out confirmed suitability of the compounds according to the invention for the sensitive and specific detection of enzymatic activity specific to ovarian cancer and, by the same, their suitability also for the specific diagnosis of ovarian cancer, and as a diagnostic marker for ovarian cancer. The mechanism of action of the compounds according to the invention consists in their specific enzymatic cleavage at the position that leads to the release of free chromophore molecules, which generates a measurable optical signal that can be used for diagnostic purposes, in particular in the diagnosis of ovarian cancer according to the present invention.
SEQUENCE LISTING IN WIPO STANDARD ST.25 FORMAT
<110> URTESTE S.A
<120> Novel diagnostic marker for ovarian cancer
<130> 17P51015PL00
<160> 1
<170> BiSSAP 1.3.6
<210> 1
<211> 4
<212> PRT
<213> Homo sapiens
<400> 1
Asp Thr Phe He
1
SEQUENCE LISTING IN WIPO STANDARD ST.26 FORMAT
<ST26SequenceListing dtd Version- ' Vl_3" fileName=" 17P51015PL00 Wykaz sekwencji.xml" softwareName- 'WIPO
Sequence" softwareVersion="2.1.1" productionDate="2022-09-26">
<ApplicantFileReference> 17P51015PL00</ApplicantFileReference>
<ApplicantName languageCode="pl">URTESTE S.A.</ApplicantName>
<ApplicantNameLatin>URTESTE S.A.</ApplicantNameLatin>
<InventionTitle languageCode="pl">Zwiqzek - marker diagnostyczny raka jajnika, sposob wykrywania aktywnosci enzymatycznej, sposob diagnozowania raka jajnika, zestaw zawierajqcy taki zwiqzek oraz zastosowania takiego zwiqzku i sposob leczenia raka j aj nika</InventionTitle>
<SequenceT otalQuantity> 1 </SequenceT otalQuantity>
<SequenceData sequencelDNumber- ' 1 ">
<INSDSeq>
<IN SD Seq_length>4</IN SDSeq_length>
<IN SD Seq_moltype> AA</IN SD Seq_moltype>
<INSD Seq di vi si on>P AT </IN SD S eq di vi si on>
<IN SD Seq_feature-table>
<INSDFeature>
<IN SDF eature_key>source</IN SDF eature_key>
<INSDF eature l ocati on> 1..4</IN SDF eature l ocati on>
<INSDF eature qual s>
<INSDQualifier>
<INSDQualifier_name>mol_type</INSDQualifier_name>
<INSDQualifier_value>protein</INSDQualifier_value>
</INSDQualifier>
<INSDQualifier id="q2">
<INSDQualifier_name>organism</INSDQualifier_name>
<INSDQualifier_value>Homo sapiens</INSDQualifier_value>
</IN SDQualifier>
</IN SDFeature qual s>
</INSDFeature>
</IN SD Seq_feature-table>
<INSDSeq_sequence>DTFI</INSDSeq_sequence>
</INSDSeq>
</SequenceData>
</S T26 S equenceLi sting>
Claims
1. A compound having formula 1 :
Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1), wherein XI comprises or consists of molecule Cl and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into the fragments Xl-Asp-Thr- Phe-Ile-OH (fragment 1) and X2 (fragment 2) with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
2. The compound according to claim 1, which compound undergoes hydrolytic cleavage, preferably proteolytic.
3. The compound according to claim 1 or 2, in which compound the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobeznoic acid (ABZ)/5-amino-2-nitrobenzoic acid (ANB), (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO2), preferably the pair of Cl and C2 is ABZ/pNA or ABZ/ANB-NH2.
4. The compound according to any one of claims 1 to 3, which compound is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (formula 2) or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3).
5. The compound according to claim 4, which compound undergoes hydrolytic cleavage with the generation of the following fragment 1 : ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2.
6. An n vitro method for detecting enzymatic activity present in a subject’s body fluid, in particular deriving from ovarian cancer cells, comprising: a) contacting the body fluid sample with the compound having formula 1 : Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1), wherein XI comprises or consists of molecule Cl and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor,
and wherein the compound undergoes enzymatic cleavage into the fragments Xl-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), and b) detecting a measurable optical signal which is generated upon spatial separation of molecules Cl and C2.
7. The in vitro method according to claim 6, wherein the enzymatic activity is hydrolytic activity, preferably proteolytic activity.
8. The in vitro method according to claim 6 or 7, wherein as the said compound the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NTL (formula 2) or the compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is used.
9. The method according to any one of claims 6 to 8, wherein as the said body fluid urine, preferably human urine, is used.
10. An in vitro method for diagnosis of ovarian cancer, wherein the presence or absence of ovarian cancer in a subject is detected by measuring enzymatic activity specific to ovarian cancer in a body fluid sample from the examined subject, and wherein the absence of the said enzymatic activity indicates the absence of ovarian cancer whereas the presence of the said enzymatic activity indicates the presence of ovarian cancer.
11. The method according to claim 10, wherein the detection of enzymatic activity is carried out by the method as defined in any one of claims 6 to 9.
12. The method according to claim 10 or 11, wherein the measurement of the said enzymatic activity is performed using the compound having formula 1 :
Xl1-Asp2-Thr3-Phe4-Ile5-X26 (formula 1), wherein XI comprises or consists of molecule Cl and X2 comprises or consists of molecule C2, wherein the pair of molecules Cl and C2 is a pair of fluorescence donor and fluorescence acceptor, and wherein the said compound undergoes enzymatic cleavage into the fragments Xl-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) with the generation of a measurable optical signal upon spatial separation of molecules Cl and C2.
13. The method according to any one of claims 10 to 12, wherein the said body fluid sample is incubated with the said compound in a measurement buffer having neutral or alkaline pH, preferably physiological, within the range of sample-to-measurement buffer ratio of 1 :2 to 1 : 10, preferably 1 :5.
14. The method according to any one of claims 10 to 13, wherein the said compound is used at a concentration of 0.1-10 mg/mL, in particular 0.25-7.5 mg/mL.
15. The method according to claims 10 to 14, wherein as the said compound the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFB (formula 2) or the compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is used.
16. The method according to any one of claims 10 to 15, wherein as the said sample a urine sample, preferably human urine, is used.
17. The method according to any one of claims 10 to 16, wherein the measurement of the said enzymatic activity comprises the measurement of absorbance intensity in the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, during 40-60 minutes, at a temperature within the range of 25-40° C, preferably 36-38° C.
18. A kit comprising the compound as defined in any one of claims 1 to 5 and a measurement buffer.
19. The kit according to claim 18, wherein the compound is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFB (formula 2) or the compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3).
20. Use of the compound as defined in any one of claims 1 to 5 for the detection of enzymatic activity specific to ovarian cancer.
21. Use of the compound as defined in any one of claims 1 to 5 for the diagnosis of ovarian cancer.
22. Use according to claim 21, wherein the diagnosis of ovarian cancer comprises the detection of primary ovarian cancer, detection of Minimal Residual Disease after surgical resection of ovarian cancer and/or detection of ovarian cancer recurrence.
23. Use according to any one of claims 21 to 22, wherein the compound is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFB (formula 2) or the compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3).
24. The compound as defined in any one of claims 1 to 5 for use as a diagnostic marker for the detection of ovarian cancer.
25. The compound for use according to claim 24, which compound is the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NFB (formula 2) or the compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3).
26. A method for the treatment of ovarian cancer, wherein
a) the presence of enzymatic activity specific to ovarian cancer is detected by the method as defined in any one of claims 6 to 9 in a body fluid sample from the examined subject and, b) if the presence of the said enzymatic activity is found in the said sample, a treatment of ovarian cancer is applied in the subject.
27. The method according to claim 26, wherein after the end of the treatment in accordance with point b), the said enzymatic activity specific to ovarian cancer is monitored at predetermined time intervals.
28. The method according to claim 26 or 27, characterized in that as the sample a urine sample, preferably human urine, is used.
29. The method according to any one of claims 26 to 28, wherein as the said compound the compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NTL (formula 2) or the compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (formula 3) is used.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PLP.442379 | 2022-09-28 | ||
| PL442379A PL442379A1 (en) | 2022-09-28 | 2022-09-28 | Ovarian cancer diagnostic marker compound, method for detecting enzymatic activity, method for diagnosing ovarian cancer, kit containing such compound, and uses of such compound and method for treating ovarian cancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2024072241A1 true WO2024072241A1 (en) | 2024-04-04 |
| WO2024072241A4 WO2024072241A4 (en) | 2024-06-06 |
Family
ID=89076258
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/PL2023/050081 WO2024072241A1 (en) | 2022-09-28 | 2023-09-27 | Compound - diagnostic marker for ovarian cancer, method for detecting enzymatic activity, method for diagnosis of ovarian cancer, kit comprising the compound, uses of the compound and method for the treatment of ovarian cancer |
Country Status (2)
| Country | Link |
|---|---|
| PL (1) | PL442379A1 (en) |
| WO (1) | WO2024072241A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187418A1 (en) * | 2017-04-04 | 2018-10-11 | Genentech, Inc. | Substrates recognized by fibroblast activation protein (fap) and methods of using the same |
| WO2020260309A1 (en) * | 2019-06-24 | 2020-12-30 | Urteste Sp. Z O.O. | Novel diagnostic marker for pancreatic cancer |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009099561A2 (en) * | 2008-01-31 | 2009-08-13 | The Brigham And Womens' Hospital, Inc. | Urinary ca125 peptides as biomarkers of ovarian cancer |
| JP7313005B2 (en) * | 2019-06-25 | 2023-07-24 | 医療法人三栄会 | Cancer biomarkers and methods for determining cancer development |
-
2022
- 2022-09-28 PL PL442379A patent/PL442379A1/en unknown
-
2023
- 2023-09-27 WO PCT/PL2023/050081 patent/WO2024072241A1/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018187418A1 (en) * | 2017-04-04 | 2018-10-11 | Genentech, Inc. | Substrates recognized by fibroblast activation protein (fap) and methods of using the same |
| WO2020260309A1 (en) * | 2019-06-24 | 2020-12-30 | Urteste Sp. Z O.O. | Novel diagnostic marker for pancreatic cancer |
Non-Patent Citations (2)
| Title |
|---|
| HAI-YU HU ET AL: "FRET-based and other fluorescent proteinase probes", BIOTECHNOLOGY JOURNAL, WILEY-VCH VERLAG, WEINHEIM, DE, vol. 9, no. 2, 27 January 2014 (2014-01-27), pages 266 - 281, XP072403285, ISSN: 1860-6768, DOI: 10.1002/BIOT.201300201 * |
| JUGNIOT NATACHA ET AL: "Neutrophil Elastase Activity Imaging: Recent Approaches in the Design and Applications of Activity-Based Probes and Substrate-Based Probes", CONTRAST MEDIA & MOLECULAR IMAGING, vol. 2019, 12 June 2019 (2019-06-12), GB, pages 1 - 12, XP093090140, ISSN: 1555-4309, DOI: 10.1155/2019/7417192 * |
Also Published As
| Publication number | Publication date |
|---|---|
| PL442379A1 (en) | 2024-04-02 |
| WO2024072241A4 (en) | 2024-06-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0988394B1 (en) | Compositions for the detection of enzyme activity in biological samples and methods of use thereof | |
| WO2012106671A1 (en) | Psa capture agents, compositions, methods and preparation thereof | |
| JP2023528638A (en) | Novel diagnostic marker for prostate cancer | |
| WO2024072241A1 (en) | Compound - diagnostic marker for ovarian cancer, method for detecting enzymatic activity, method for diagnosis of ovarian cancer, kit comprising the compound, uses of the compound and method for the treatment of ovarian cancer | |
| US20240353410A1 (en) | Fret enzymatic substrate and uses thereof in liver cancer | |
| AU2023256464A1 (en) | Compound - diagnostic marker for kidney cancer, method lor detecting enzymaticactivity, method for diagnosis of kidney cancer, kit comprising the compound, uses of the compound and method for the treatment of kidney cancer | |
| WO2024005658A1 (en) | Compound-diagnostic marker for uterine body cancer, method for detecting enzymatic activity, method for diagnosis of uterine body cancer, kit comprising the compound, uses of the compound and method for the treatment of uterine body cancer | |
| WO2024151174A2 (en) | Compound - diagnostic marker for biliary tract cancer, method for detecting enzymatic activity, method for diagnosis of biliary tract cancer, kit comprising the compound, uses of the compound and method for the treatment of biliary tract cancer | |
| AU2023241522A1 (en) | Compound - diagnostic marker for intestinal cancer, method for detecting enzymatic activity, method for diagnosis of intestinal cancer, kit comprising the compound, uses of the compound and method for the treatment of intestinal cancer | |
| AU2022407963A1 (en) | Fret enzymatic substrate and uses thereof in lung cancer. | |
| WO2024177524A1 (en) | Compound, diagnostic marker for stomach cancer, method for detecting enzymatic activity, method for the diagnosis of stomach cancer, kit comprising the compound, uses of the compound and method for the treatment of stomach cancer | |
| JP5170407B2 (en) | Diabetes complication test reagent | |
| EP3431490B1 (en) | Chemical compounds for use as diagnostic markers of inflammation and neoplams, the method for synthesis of the chemical compounds, diagnostic kit for use in diagnosis of inflammatory processes and epithelial neoplasms and in vitro diagnostic method of inflammatory processes and epithelial neoplasms | |
| WO2020017984A1 (en) | Chemical compounds for use as diagnostic markers of inflammation and neoplasms | |
| PL231469B1 (en) | New compound, method for obtaining it, pharmaceutical solution containing the new compound, method for determination of the presence of tumor disease, accessories for cancer detection and application of the hydrolysis of the new compound for cancer detection | |
| JP5278873B2 (en) | Cancer diagnostic reagents | |
| EP4139470A1 (en) | Cancer-related activity sensors | |
| PL238699B1 (en) | Chemical compound used as a diagnostic marker of cancer, method of its preparation, method of diagnosing epithelial cancers | |
| PL238700B1 (en) | Chemical compounds used as diagnostic markers of inflammation and cancer to be used as a diagnostic kit, and method of diagnosing and differentiating inflammatory processes and epithelial cancers | |
| PL241174B1 (en) | Chemical compound - diagnostic marker of pancreatic cancer, method of its production and use in cancer diagnostics | |
| PL238575B1 (en) | New compound, method for obtaining it, a kit containing the new compound and application of the new compound for detection of epithelial tumors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23817848 Country of ref document: EP Kind code of ref document: A1 |